Project Summary Chronic liver disease is becoming a major public health issue with estimates of up to 40% prevalence of non- alcoholic fatty liver disease (NAFLD) and 12% with non-alcoholic steatohepatitis (NASH) in the U.S. Liver disease and cirrhosis was the 12th leading cause of mortality in the U.S. in 2014. Obesity and metabolic syndrome are important causes of fatty liver disease or steatosis, with NAFLD rates of up to 70% in type 2 diabetic patients, and 30%-90% of those with obesity. It is also affecting children. NASH and liver fibrosis can lead to organ failure. The estimated cost of NAFLD alone in the U.S., in direct medical costs are about $103 billion. There are currently no drugs available to treat either steatosis or liver fibrosis. In 2009 it was shown that the matricellular signaling protein CCN3 is a natural regulator, or balancer, of the well- established pro-fibrotic protein and family member CCN2. The anti-fibrotic role of CCN3 and potential as a drug was recently shown in treatment models of renal fibrosis driven by diabetes and obesity, and in models of skin scarring. In the former, it showed the capacity to halt and even reverse established fibrosis. To create a more effective drug, BLR Bio in a peptide discovery program, identified two critical regions on the CCN3 protein responsible for this actively and has proposed stable synthetic peptides. Our long-term goal is to use these improved drug forms to treat NAFLD, both reducing the mortality and morbidity associated the disease, and dramatically curbing the growing healthcare costs. Our preliminary experiments were carried out in a model of type 2 diabetes and obesity, a model we used to study renal fibrosis. As part of those studies, we generated intriguing data suggesting that our lead peptide, BLR200 (BLR100 has not yet been tested), is able to block in a dose-dependent manner the upregulation of key selected fibrosis genes in the liver, and the early manifestation of liver fibrosis. Surprisingly, it also blocked and perhaps reversed steatosis in almost half of the animals. In collaboration with liver experts at the University of Michigan, we aim to confirm proof of principle in animals. Specifically, we will test and compare BLR100 and BLR200, for dose-response efficacy in the FATZO rodent model, which on a high fat diet develops NAFLD and NASH-like pathology. We will define the two peptides? ability to block, and perhaps reverse, development of established liver injury (steatosis and its down-stream consequences including inflammation, hepatocyte necrosis and fibrosis [NASH]). Controls will include mice on a normal chow diet and placebo treated mice on the high fat diet. Parameter?s tested will be aligned to currently accepted clinical measures of progression including biomarkers. Added will be a proteomic analysis to begin elucidating the pathways involved. Completion of these milestones will set the stage for a Phase 2 STTR grant and partner investment to complete our preclinical program and to open an IND, the 2nd significant milestone to the commercialization of a novel and much needed treatment.